1. Field of the Invention
The present invention relates to novel parathyroid hormone peptide (PTH) derivatives and to novel parathyroid hormone-related peptide (PTHrP) derivatives. In particular, the invention relates to PTH and PTHrP minimized peptide and derivatives thereof that still retain biological activity.
2. Description of Related Art
Parathyroid hormone (PTH) is a major regulator of calcium homeostasis whose principal target cells occur in bone and kidney. Regulation of calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone indirectly by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D. PTH exerts these effects primarily through receptor-mediated activation of adenylate cyclase and phospholipase C.
Disruption of calcium homeostasis may produce many clinical disorders (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a lesion (e.g., adenoma, hyperplasia, or carcinoma) of the parathyroid glands. Another type of hypercalcemia, humoral hypercalcemia ofmalignancy (HHM) is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues. PTHrP is normally found at low levels in many tissues, including keratinocytes, brain, pituitary, parathyroid, adrenal cortex, medulla, fetal liver, osteoblast-like cells, and lactating mammary tissues. In many HHM malignancies, PTHrP is found in the circulatory system at high levels, thereby producing the elevated calcium levels associated with HHM.
The pharmacological profiles of PTH and PTHrP are nearly identical in most in vitro assay systems, and elevated blood levels of PTH (i.e., primary hyperparathyroidism) or PTHrP (i. e., HHM) have comparable effects on mineral ion homeostasis (Broadus, A. E. and Stewart, A. F., xe2x80x9cParathyroid hormone-related protein: Structure, processing and physiological actions,xe2x80x9d in Basic and Clinical Concepts, Bilzikian, J. P. et al., eds., Raven Press, New York (1994), pp.259-294; Kronenberg, H. M. et al., xe2x80x9cParathyroid hormone: Biosynthesis, secretion, chemistry and action,xe2x80x9d in Handbook of Experimental Pharmacology, Mundy, G. R. and Martin, T. J., eds., Springer-Verlag, Heidelberg (1993), pp. 185-201). The similarities in the biological activities of the two ligands can be explained by their interaction with a common receptor, the PTH/PTHrP receptor, which is expressed abundantly in bone and kidney (Urena, P. et al., Endocrinology 134:451-456 (1994)).
Native human parathyroid hormone is an unmodified polypeptide of 84 amino acids. It is secreted from the parathyroid glands in response to low blood calcium levels and acts on osteoblast (bone-building cells) in bone, and on tubular epithelial cells of kidney. The hormone interacts with a cell surface receptor molecule, called the PTH-1 receptor or PTH/PTHrP receptor, which is expressed by both osteoblast and renal tubular cells. PTHrP, the major cause of the humoral hypercalcemia of malignancy, also has normal functions that include roles in development. PTHrP has 141 amino acids, though variants also occur that result from alternative gene splicing mechanisms. PTHrP plays a key role in the formation of the skeleton through a process that also involves binding to the PTH-1 receptor (Karaplis, A. C., et al., Genes and Dev. 8:277-289 (1994) and Lanske, B., et al., Science 273:663-666 (1996)).
The PTH-1 receptor is homologous in primary structure to a number of other receptors that bind peptide hormones, such as secretin (Ishihara, T. et al., EMBO J. 10:1635-1641 (1991)), calcitonin (Lin, H. Y. et al., Science 254:1022-1024 (1991)) and glucagon (Jelinek, L. J. et al., Science 259:1614-1616 (1993)); together these receptors form a distinct family called receptor family B (Kolakowski, L. F., Receptors and Channels2:1-7 (1994)). Within this family, the PTH-1 receptor is unique, in that it binds two peptide ligands and thereby regulates two separate biological processes. A recently identified PTH receptor subtype, called the PTH-2 receptor, binds PTH but not PTHrP (Usdin, T., et al., J. Biol. Chem. 270:15455-15458(1995)). This observation implied that structural differences in the PTH and PTHrP ligands determined selectivity for interaction with the PTH-2 receptor. The PTH-2 receptor has been detected by RNA methods in the brain, pancreas and vasculature, however, its biological function has not been determined (Usdin, T., et al., J. Biol. Chem. 270:15455-15458 (1995)). It is hypothesized that the family B receptors use a common molecular mechanism to engage their own cognate peptide hormone (Bergwitz, C., et al., J. Biol. Chem. 271:26469-26472 (1996)).
The binding of either radio labeled PTH(1-34) or PTHrP(1-36) to the PTH-1 receptor is competitively inhibited by either unlabeled ligand (Jxc3xcppner, H. et al., J. Biol. Chem. 263:8557-8560 (1988); Nissenson, R. A. et al., J. Biol. Chem. 263:12866-12871 (1988)). Thus, the recognition sites for the two ligands in the PTH-1 receptor probably overlap. In both PTH and PTHrP, the 15-34 region contains the principal determinants for binding to the PTH-1 receptor. Although these regions show only minimal sequence homology (only 3 amino acid identities), each 15-34 peptide can block the binding of either PTH(1-34) or PTHrP(1-34) to the PTH-1 receptor (Nussbaum, S. R. et al., J. Biol. Chem. 255:10183-10187 (1980); Caulfield, M. P. et al., Endocrinology 127:83-87 (1990); Abou-Samra, A.-B. et al., Endocrinology 125:2215-2217 (1989)). Further, the amino terminal portion of each ligand is required for bioactivity, and these probably interact with the PTH-1 receptor in similar ways, since 8 of 13 of these residues are identical in PTH and PTHrP.
Both PTH and PTHrP bind to the PTH-1 receptor with affinity in the nM range; the ligand-occupied receptor transmits a xe2x80x9csignalxe2x80x9d across the cell membrane to intracellular effector enzymes through a mechanism that involves intermediary heterotrimeric GTP-binding proteins (G proteins). The primary intracellular effector enzyme activated by the PTH-1 receptor in response to PTH or PTHrP is adenylyl cyclase (AC). Thus, PTH induces a robust increase in the xe2x80x9csecond messengerxe2x80x9d molecule, cyclic adenosine monophosphate (cAMP) which goes on to regulate the poorly characterized xe2x80x9cdownstreamxe2x80x9d cellular processes involved in bone-remodeling (both bone formation and bone resorption processes). In certain cell-based assay systems, PTH can stimulate effector enzymes other than AC, including phospholipase C (PLC), which results in production of inositol triphosphate (IP3), diacylglycerol (DAG) and intracellular calcium (iCa2+). The roles of these non-cAMP second messenger molecules in bone metabolism are presently unknown.
Osteoporosis is a potentially crippling skeletal disease observed in a substantial portion of the senior adult population, in pregnant women and even in juveniles. The disease is marked by diminished bone mass, decreased bone mineral density (BMD), decreased bone strength and an increased risk of bone fracture. At present, there is no effective cure for osteoporosis, though estrogen, calcitonin and the bisphosphonates, etidronate and alendronate are used to treat the disease with varying levels of success through their action to decrease bone resorption. Since parathyroid hormone regulates blood calcium and the phosphate levels, and has potent anabolic (bone-forming) effects on the skeleton, in animals (Shen, V., e al., Calcif Tissue Int. 50:214-220 (1992); Whitefild, J. F., et al., Calcif Tissue Int. 56:227-231 (1995) and Whitfield, J. F., et al., Calcif. Tissue Int. 60:26-29 (1997)) and humans (Slovik, D. M., et al., J. Bone Miner. Res. 1:377-381 (1986); Dempster, D. W., et al., Endocr. Rev. 14:690-709 (1993) and Dempster, D. W., et al., Endocr. Rev. 15:261 (1994)) when administered intermittently, PTH, or PTH derivatives, are prime candidates for new and effective therapies for osteoporosis.
Truncated PTH derivatives such as PTH(1-34) and PTH(1-31) are active in most assay systems and promote bone-formation (Whitefild, J.F., et al., Calcif. Tissue Int. 56:227-231 (1995); Whitfield, J. F., et al., Calcif Tissue Int. 60:26-29 (1997); Slovik, D. M., et al., J. Bone Miner. Res. 1:377-381 (1986); Tregear, G. W., et al., Endocrinology 93:1349-1353 (1973); Rixon, R. H., et al., J. Bone Miner. Res. 9:1179-1189 (1994); Whitfield, J. F. and Morley, P., Trends Pharmacol. Sci. 16:372-386 (1995) and Whitfield, J. F., et al., Calcif. Tissue Int. 58:81-87(1996)). Butthese peptides are still too large for efficient non-parenteral delivery and low cost. The discovery of an even smaller xe2x80x9cminimizedxe2x80x9d version of PTH or PTHrP would be an important advance in the effort to develop new treatments for osteoporosis.
PTH and PTHrP derivatives that have amino acid substitutions or deletions in the 1-14 region usually exhibit diminished activity (Tregear, G. W., et al., Endocrinology 93:1349-1353 (1973); Goltzman, D., et al., J. Biol. Chem. 250:3199-3203 (1975); Horiuchi, N., et al., Science 220.1053-1055 (1983) and Gardella, T. J., et al., J. Biol. Chem. 266:13141-13146 (1991))
Several short NH2-terminal PTH or PTHrP peptides have been investigated previously, but no activity was detected. For example, bPTH(1-12) was inactive in adenylyl cyclase assays performed in rat renal membranes (Rosenblatt, M., xe2x80x9cParathyroid Hormone. Chemistry and Structure-Activity Relations,xe2x80x9d in Pathobiology Annual, Ioachim, H. L., ed., Raven Press, New York (1981), pp. 53-84) and PTHrP(1-16) was inactive in AC assays performed in Chinese hamster ovary (CHO) cells expressing the cloned rat PTH-1 receptor (Azurani, A., et al., J. Biol. Chem. 271:14931-14936 (1996)). It has been known that residues in the 15-34 domain of PTH contribute importantly to receptor binding affinity, as the PTH(15-34) fragment binds weakly to the receptor, but this peptide does not activate AC (Naussbaum, S. R., et al., J. Biol. Chem. 255:10183-10187 (1980) and Gardella, T. J., et al., Endocrinology 132:2024-2030 (1993)).
The relatively large size of native PTH or PTHrP presents challenges to the use of these peptides as treatments for osteoporosis. In general, a protein of this size is not suitable for use as a drug, since it cannot be delivered effectively by simple methods such as nasal inhalation. Instead, injection is required, and in the case of PTH, daily, or almost daily injections would most likely be needed to achieve increases in bone formation rates. Additionally, larger peptides are technically difficult and expensive to prepare by conventional synthetic chemistry methods. Alternative methods employing recombinant DNA and cell-based expression systems are also expensive, potentially vulnerable to contamination by foreign proteins and do not circumvent the delivery problem.
Accordingly, it would be advantageous for those skilled in the art to be able to identify a small molecule analog (either peptide or non-peptide) that is based on the larger peptide and yet which still retains the desired biological activities. The activity may at first be weak relative to the intact peptide, but further optimization can lead to enhanced efficacy and potency.
The present invention relates to PTH(1-14)/PTHrP(1-14) peptides and derivatives thereof Compounds of the invention which include PTH(1-14)/PTHrP(1-14) peptides, fragments thereof, derivatives thereof, pharmaceutically acceptable salts thereof, and N- or C-derivatives thereof, are hereinafter collectively referred to as xe2x80x9ccompounds of SEQ ID NO:1 and derivatives thereofxe2x80x9d.
In detail, the invention provides synthetic and/or recombinant biologically active peptide derivatives of PTH(1-14) and PTHrP(1-14). In one specific embodiment, this invention provides a biologically active peptide at least 85% identical to a peptide consisting essentially of the formula:
(a) X01ValSerGluX02GlnLeuX03HisX04X05GlyLysX06(SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-9, 1-10, 1-11,1-12, or 1-13;
(c) pharmaceutically acceptable salts thereof; or
(d) N- or C-derivatives thereof;
wherein:
X01 is Ser or Ala;
X02 is Ile or His;
X03 is Met, Leu or Nle;
X04 is Asn or Asp;
X05 is Leu or Lys; and
X06 is His or Ser, provided that said peptide is not PTHrP(1-14).
In accordance with yet a further aspect of the invention, this invention also provides pharmaceutical compositions comprising
(a) a biologically active peptide at least 85% identical to a peptide consisting essentially of the formula: SerValSerGluIleGlnLeuMetHisAsnLeu GlyLysHis (SEQ ID NO:3);
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
(c) pharmaceutically acceptable salts thereof; or
(d) N- or C-derivatives thereof; and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect of the invention, this invention also provides pharmaceutical compositions comprising
(a) a biologically active peptide at least 85% identical to a peptide consisting essentially of the formula: AlaValSerGluHisGlnLeuLeuHisAspLys GlyLysSer (SEQ ID NO:4);
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
(c) pharmaceutically acceptable salts thereof; or
(d) N- or C-derivatives thereof; and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect of the invention, this invention provides a nucleic acid molecule consisting essentially of a polynucleotide encoding a biologically active peptide which has an amino acid sequence selected from the group consisting of:
(a) X01ValSerGluX02GlnLeuX03HisX04X05GlyLysX06(SEQ ID NO:1); or
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
wherein:
X01 is Ser or Ala;
X02 is Ile or His;
X03 is Met, Leu or Nle;
X04 is Asn or Asp;
X05 is Leu or Lys; and
X06 is His or Ser,
provided that said peptide is not PTHrP(1-14).
In accordance with yet a further aspect of the invention, this invention provides a recombinant DNA molecule comprising: (1) an expression control region, said region in operable linkage with (2) a polynucleotide sequence coding for a biologically active peptide, wherein said peptide is selected from the group consisting of:
(a) X01ValSerGluX02GlnLeuX03HisX04X5GlyLysX06(SEQ ID NO:1); or
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
wherein:
X01 is Ser or Ala;
X02 is Ile or His;
X03 is Met, Leu or Nle;
X04 is Asn or Asp;
X05 is Leu or Lys; and
X06 is His or Ser,
provided that said peptide is not PTHrP(1-14).
In accordance with yet a further aspect of the invention, this invention provides a method for treating mammalian conditions characterized by decreases in bone mass, which method comprises administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active peptide, wherein said peptide comprises an amino acid sequence at least 85% identical to a member selected from the group consisting essentially of:
(a) X01ValSerGluX02GlnLeuX03HisX04X05GlyLysX06(SEQ ID NO:1);
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
(c) pharmaceutically acceptable salts thereof; or
(d) N- or C-derivatives thereof;
wherein:
X01 is Ser or Ala;
X02 is Ile or His;
X03 is Met, Leu or Nle;
X04 is Asn or Asp;
X05 is Leu or Lys; and
X06 is His or Ser,
provided that said peptide is not PTHrP(1-14); and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect of the invention, this invention provides a method for the treatment of a patient having need of a biologically active peptide comprising administering a therapeutically effective amount of a peptide, wherein said peptide comprises an amino acid sequence at least 85% identical to a member selected from the group consisting essentially of:
(a) a biologically active peptide consisting essentially of the formula: SerValSerGlulleGlnLeuMetHisAsnLeuGlyLysHis (SEQ ID NO:3);
(b) fragments thereofcontaining amino acids 1-9,1-10, 1-11, 1-12, or 1-13;
(c) N- or C-derivatives thereof; or
(d) pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect of the invention, this invention provides a method for the treatment of a patient having need of a biologically active peptide comprising administering a therapeutically effective amount of a peptide, wherein said peptide comprises an amino acid sequence at least 85% identical to a member selected from the group consisting essentially of:
(a) a biologically active peptide consisting essentially of the formula: AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSer (SEQ ID NO:4);
(b) fragments thereof containing amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
(c) N- or C-derivatives thereof, or
(d) pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect of the invention, there is provided a method for treating a medical disorder that results from altered or excessive action of the PTH-1/PTH-2 receptor, comprising administering to a patient a therapeutically effective amount of a biologically active peptide wherein said peptide comprises an amino acid sequence at least 85% identical to a member selected from the group consisting essentially of:
(a) X01ValSerGluX02GlnLeuX03HisX04X05GlyLysX06(SEQ ID NO:1);
(b) fragments thereofcontaining amino acids 1-9, 1-10, 1-11, 1-12, or 1-13;
(c) pharmaceutically acceptable salts thereof; or
(d) N- or C-derivatives thereof,
wherein:
X01 is Ser or Ala;
X02 is Ile or His;
X03 is Met, Leu or Nle;
X04 is Asn or Asp;
X05 is Leu or Lys; and
X06 is His or Ser,
provided said peptide is not PTHrP(1-14); and a pharmaceutically acceptable carrier sufficient to inhibit activation of the PTH-1/PTH-2 receptor of said patient.
In accordance with yet a further aspect of the invention, this invention also provides a method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of a labeled peptide of SEQ ID NO: 1 or a derivative thereof and determining the uptake of said peptide into the bone of said patient. The peptide may be labeled with a label selected from the group consisting of: radio label, flourescent label, bioluminescent label, or chemiluminescent label. An example of a suitable radio label is 99m Tc.
In accordance with yet a further aspect of the invention, any amino-acid substitutions at positions 1-9, and more particularly those amino acid substitutions at amino acid positions 10, 11, 12, 13, and/or 14, which do not destroy the biological activity of the PTH(1-14)/PTHrP(1-14) peptide analog to antagonize or agonize the PTH-1/PTH-2 receptor (as determined by assays known to the skilled artisan and discussed below), are also included within the scope of the present invention.